The long term goals of the proposed study are (1) to understand the biochemical mechanisms underlying protein targeting and biogenesis and, (2) to reveal the evolutionary origin and development of the hydrogenosome, and organelle found in the human-infective parasite Trichmonas vaginalis. This organelle plays a central role in carbohydrate metabolism and drug susceptibility of T. vaginalsi - a common etiologic agent of vaginitis. Hydrogenosomes provide a target for the selective killing of this parasite, as the organelle is not found in the human host. Thus, in addition to their biological merit, these studies provide a framework for future medical applications. We have developed both in vitro and in vivo protein import assays to allow the detailed study of the mechanisms used to accurately target and translate proteins into hydrogenosomes. Using these assays and direct biochemical approaches to characterize hydrogenosomal membrane proteins, the applicant have found that the general mechanisms used to target proteins to the matrix of hydrogenosomes resemble those used for mitochondrial proteins, although distinct differences exist. We have also found that homologues of mitochondrial proteins are present in hyfrogensomes. These data strongly argue for a connon evolutionary origin for hydrogenosomes and mitochodria. To extend and changgenge this hypothesis, the applicant proposes to (1) identify major membrane proteins and putative translocation receptors of hydrogenosomes and to determine their functional and evolutionary relationship with mitochondrial proteins, (2) identify essential signals necessary to target proteins into either the matricx or the membranes of the hydrogensome, (3) develop mutagensis and functional complelemetation asays to identify and character proteins involved in hydrogenosomal biogenesis, and (4) identify cytosolic proteins that play an essential role in targeting and translocating proteins into the organelle. This detailed analysis will provide valuable insights into the origin and biogenesis of hydrogenosomes and information of potential chemotherapeutic importance.